Adenosine deaminase inhibitor and novel Bacillus sp. Iada-7 strain which produces it

ABSTRACT

The present invention relates to an adenosine deaminase inhibitor and a novel  Bacillus  sp. strain which produces it. Particularly, the present 5 invention relates to the adenosine deaminase inhibitor, and the novel  Bacillus  sp. IADA-7 producing the above adenosine deaminase inhibitor. The adenosine deaminase inhibitor of the present Invention shows superior antibacterial and anticancer activities to the previously reported adenosine deaminase inhibitors.

This application is a 371 of PCT/KR2004/000652 filed on Mar. 24, 2004,published on Oct. 7, 2004 under publication number WO 2004/085410 A1which claims priority benefits from South Korean Patent ApplicationNumber 10-2003-0019238 filed Mar. 27, 2003.

FIELD OF THE INVENTION

The present invention relates to an adenosine deaminase inhibitor and anovel Bacillus sp. strain producing the same. Particularly, the presentinvention relates to the adenosine deaminase inhibitor of Formula 1 andthe novel Bacillus sp. IADA-7 (KCTC 10446BP) producing the aboveadenosine deaminase inhibitor:

wherein R₁ and R₂ are H or a C₁˜C₁₀ alkyl group, respectively.

BACKGROUND OF THE INVENTION

Adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4), one of theenzymes involved in purine metabolism, generates inosine and ammonia byremoving an amino group coupled to the sixth carbon of adenosine and ispresent ubiquitously in nature.

Purine is synthesized via two pathways: a de novo pathway from amicromolecule precursor and a salvage pathway from the purine.

The salvage pathway is a process which reuses a foreign substance suchas a degrading product generated by destroying unstable RNA within acell, a nucleic acid of dead cell and a degrading product ofnucleotides, and has the advantage of preventing a loss in vital energyand precursors. While the de novo pathway is preserved in all species,the salvage pathway varies depending on the kind of species.

It has been known that animals, plants and microorganisms have aspecific inhibitor for each enzyme reaction and these inhibitors aremostly macromolecule peptides, but an inhibitor for the adenosinedeaminase is generally an adenosine analogue which is a micromoleculecompound having 500 Da or less of a molecular weight. Further, it isknown that an enzyme inhibitor isolated from a microbial metabolite is amicromolecular substance having an extremely low toxicity with a newstructure. Some inhibitors are very similar in their structures. Thepharmaceutical composition of the present invention substrates, whileothers are completely different from their substrates.

As shown in the biosynthesis of antibiotics, there are cases that aplasmid is involved in a characteristic part of the procedure forbiosynthesizing an enzyme inhibitor.

There have been reports on several microorganism-originated inhibitorsfor adenosine deaminase, such as coformycin(3-(α-D-ribo-furanosyl)-6,7,8-trihydroimidazol(1,3)diazepin-8(R)-ol)produced by Streptomyces kaniharaensis SF-557; and cordycepsin and2′-deoxy coformycin produced by Aspergillus nidulans Y-176-2.

Coformycin produced by Streptomyces which producesformycin(7-amino-3-(β-ribofuranosyl)pyrazolo(4,3-d)pyrimidine) is aspecific inhibitor for the adenosine deaminase and its inhibition is incompetition with a substrate. Further, coformycin, together withformycin, shows a synergistic effect in inhibiting a bacterial growth inmost bacteria except for Xanthomonas oryzae, and effectively inhibits aproliferation of Yoshida rat sarcoma cells.

Further, it has been reported 9-α-D-mannopyranosyladenine(1),9-β-D-xylopyranosyladenine(2), 9-α-D-arabinopyranosyladenine(3),9-α-L-rhamnopyranosyladenine(4), 9-β-D-fucopyranosyl adenine(5),9-β-L-fucopyranosyladenine(6) as adenosine deaminase inhibitors. All ofthem except for 9-α-D-mannopyranosyladenine(4) act as competitiveinhibitors, and 9-α-L-rhamnopyranosyladenine is known to have thestrongest inhibitory effect.

It has been known that cytotoxicity is developed in a cell inhibited byerythro-9-(2-hydroxy-3-nonyl)adenine), an inhibitor of an adenosinedeaminase, when the cell is treated with adenosine and deoxyadenosine.

It has been also reported that injection of an adenosine analogue suchas arabinosyladenine, codycepin or formycin as into an animal canincrease an anti-cancer effect.

An enzyme inhibitor has been effectively used for analyzing humanphysiological functions and medically important pathological phenomena,and in particular, specific inhibitors have been known useful forbiochemical analyses of biological functions or pathogens. Further, theenzyme inhibitors are powerful means in discovering variouscharacteristics of enzymes such as active sites of enzymes, in vivoroles and physiological functions, and they can be also applicable asmarkers for diagnosing various diseases or as therapeutic agents.

The study on adenosine deaminase has been conducted since 1980s when aninhibitor of adenosine deaminase was known as an inhibitor of an immunesystem based on the discovery that the deficiency in adenosinedeaminase, an essential factor involved in immune system, leads to thedecrease in T-lymphocyte and B-lymphocyte thus resulting in animmunodeficiency.

Further, it has been found that the adenosine deaminase inhibitorincreases the amount of ATP synthesis in petroleum-decomposing yeastusing adenosine as a substrate, and thus its application to ATPsynthesis has been proposed.

The studies about enzyme inhibitors have been actively carried out atnumerous research institutions. As a result, new metabolic systems orenzyme systems have been discovered, thus clarifying the controllingrelationships among biophysical functions.

Most of adenosine deaminase inhibitors reported till now have beengrouped to a purine analogue family, which is produced by Actinomycetes,but they are very toxic to human cells when applied for medicaltreatments, and therefore, it has been on urgent need to develop a newdrug applicable for clinical trials.

Further, there has been no report on adenosine deaminase inhibitorsproduced by bacterial strains.

The present inventors have isolated a new bacterial strain from a soilwhich produces an adenosine deaminase inhibitor compound and found thatthe inhibitor compound has antibacterial and anticancer activities.

SUMMARY OF THE INVENTION

Accordingly, an object of the present invention is to provide anadenosine deaminase inhibitor compound and a pharmaceutically acceptablesalt thereof.

Another object of the present invention is to provide a novel Bacillussp. IADA-7 (KCTC 10446BP) strain which produces the adenosine deaminaseinhibitor and a method for producing same.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other objects and features of the present invention willbecome apparent from the following description of the invention, whentaken in conjunction with the accompanying drawings, wherein

FIG. 1 shows an optical microphotograph of the Bacillus sp. IADA-7 (KCTC10446BP) strain of the present invention.

FIG. 2 shows a graph representing a bacterial growth, synthesis of aninhibitor compound and pH variation according to time of culturing theBacillus sp. IADA-7 (KCTC 10446BP) strain of the present invention.

FIG. 3 shows a result of purifying the inhibitor compound of the presentinvention with Dowex ion exchange resin.

FIG. 4 shows a result of purifying the inhibitor compound of the presentinvention with a gel filtration.

FIG. 5 shows a flowchart of representing a purifying procedure of theinhibitor compound of the present invention.

FIG. 6 shows a result of developing the inhibitor compound of thepresent invention on a normal phase TLC.

FIG. 7 shows a result of developing the inhibitor compound of thepresent invention on a reverse phase TLC.

FIG. 8 shows a result of loading the inhibitor compound of the presentinvention to high voltage paper electrophoresis.

FIG. 9 shows a result of analyzing the inhibitor compound of the presentinvention with HPLC.

FIG. 10 shows a graph representing the relationship between each peakheight and an injection amount in HPLC analysis of the inhibitorcompound of the present invention.

FIG. 11 shows a result of determining a molecular weight of theinhibitor compound of the present invention.

FIG. 12 shows a graph representing a competitive inhibition of theinhibitor compound of the present invention to the adenosine deaminasederived from Nocardiodes sp.

FIG. 13 shows a graph representing a competitive inhibition of theinhibitor compound of the present invention to the adenosine deaminasederived from bovine pancreas.

FIG. 14 shows UV absorption spectrum of the inhibitor compound of thepresent invention.

FIG. 15 shows IR absorption spectrum of the inhibitor compound of thepresent invention.

FIG. 16 shows ¹H-NMR spectrum of the inhibitor compound of the presentinvention.

FIG. 17 shows ¹³C-NMR spectrum of the inhibitor compound of the presentinvention.

FIG. 18 shows ¹³C-NMR spectrum of the inhibitor compound of the presentinvention.

FIG. 19 shows EI-MASS spectrum of the inhibitor compound of the presentinvention.

FIG. 20 shows a structural formula of the inhibitor compound of thepresent invention.

FIG. 21 shows a result of examining cytotoxicity of the inhibitorcompound of the present invention to a human transitional-cell carcinoma(bladder) derived from a testis.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with one aspect of the present invention, there isprovided an adenosine deaminase inhibitor compound of Formula 1 and apharmaceutically acceptable salt thereof:

wherein R₁ and R₂ are H or a C₁˜C₁₀ alkyl group, respectively.

The present invention also provides a pharmaceutical compositioncomprising the compound showing an adenosine deaminase inhibitoryactivity as an effective ingredient.

In addition, the present invention provides an antibacterial or ananticancer agent comprising a compound showing an adenosine deaminaseinhibitory activity as an effective ingredient.

Further, the present invention provides a novel Bacillus sp. IADA-7strain (KCTC 10446BP) which produces the compound of Formula 1.

Furthermore, the present invention provides a method for producing thecompound of Formula 1 which comprises the steps of culturing theBacillus sp. IADA-7 (KCTC 10446BP) strain and purifying the compoundfrom the culture solution.

Hereinafter, the present invention is described in detail.

The present invention relates to the adenosine deaminase inhibitor ofFormula 1 and the novel Bacillus sp. IADA-7 (KCTC 10446BP) producing thesame.

The bacterial strain producing the adenosine deaminase inhibitor hasbeen isolated from soil and its morphological, cultural and biochemicalfeatures have been characterized. As a result, the isolated bacterialstrain has been identified as a Bacillus sp. and designated IADA-7. TheBacillus sp. IADA-7 strain of the present invention has been depositedat Korean Collection for Type Cultures (Address: #52, Oun-dong,Yusong-ku, Taejon 305-333, Republic of Korea) on Mar. 18, 2003 andassigned with the accession number of KCTC 10446BP, in accordance withthe terms of the Budapest Treaty on the International Recognition of theDeposit of Microorganism for the Purpose of Patent Procedure.

Further, the present invention also includes a method for producing thecompound of Formula 1 by culturing the Bacillus sp. IADA-7 (KCTC10446BP) strain.

wherein R₁ and R₂ are H or a C₁˜C₁₀ alkyl group, respectively.

It has been confirmed that the compound of Formula 1 produced by theabove-mentioned method shows high antibacterial and anticanceractivities as an adenosine deaminase inhibitor.

Meanwhile, the compound of Formula 1 of the present invention may beused in a form of a pharmaceutically acceptable salt, and in particular,its acid added salt prepared by using a pharmaceutically acceptable freeacid is preferable. A pharmaceutically acceptable acid added salt of thecompound of Formula 1 may be prepared according to the conventionalmethod well-known in the art. Inorganic and organic acids may be used asthe free acid. The inorganic acid includes, but is not limited to,hydrochloric acid, bromic acid, sulfuric acid and phosphoric acid; andthe organic acid, citric acid, acetic acid, lactic acid, tartaric acid,maleic acid, fumaric acid, formic acid, propionic acid, oxalic acid,trifluoroacetic acid, benzoic acid, gluconic acid, methansulfonic acid,glycolic acid, succinic acid, 4-toluenesulfonic acid, galacturonic acid,embonic acid, glutamic acid or asparaginic acid.

Accordingly, the pharmaceutical composition comprising the compound ofFormula 1 or a pharmaceutically acceptable salt thereof as an effectiveingredient can be effectively used for developing an antibacterial or ananticancer agent as an adenosine deaminase inhibitor.

The pharmaceutical composition of the present invention can beadministered orally or via parental routes such as percutaneous,subcutaneous, intravenous or intramuscular methods, and manufactured ina form of a common medicine or a health improving food.

The pharmaceutical composition of the present invention may beformulated in the form of tablets, troches, soluble or oily suspensions,powders or granules, emulsions, hard or soft capsules, syrups orelixirs. Pharmaceutical formulations in the form of tablets and capsulesmay further comprise binding agents such as lactose, saccharose,sorbitol, manitol, starch, amylopectin, cellulose or gelatin;emulsifying agents such as dicalcium phosphate; disintegrating agentssuch as corn starch or sweet potato starch; and lubricating agents suchas magnesium stearate, calcium stearate, sodium stearate, sodiumstearylfumarate or polyglycol wax. In case of capsules, they may furthercomprise soluble carriers such as fat oil besides the above mentionedsubstances.

Further, the pharmaceutical composition may be parenterally administeredvia subcutaneous injection, intravenous injection, intramuscularinjection or chest injection. Pharmaceutical formulations for parentaladministration may be prepared by mixing toluquinol in water withstabilizing agents or buffering agents to obtain a solution or asuspension and formulating the solution or the suspension into an ampleor a vial as a unit dosage form.

For clinical administration purpose, a typical daily dose of thecompound of Formula 1 may range from 1 to 50 mg/kg body weight,preferably from 5 to 20 mg/kg body weight and can be administrated in asingle dose or in divided dose. However, it can be changed into thehigher or lower daily dose of the effective ingredient depending on adisease. Further, it should be understood that the amount of theeffective ingredient actually administrated to a certain patient oughtto be determined in light of various relevant factors including the kindof effective compound administered, body weight, age, sex, healthconditions, diet and excretion rate of an individual patient, theselected route of administration, the combination of drugs and theseriousness of the patient's symptom.

Meanwhile, the culture and identification of a bacterial strain in thepresent invention have been carried out according to the methods asdescribed below.

<Reagents and Equipments>

Bacteria were cultured in a 120Rev,×6 cm shaker and the removal ofcultured bacterial cells and preparation of a crude enzyme solution wereperformed by using H50A-6 centrifuge (Han-il, Korea). RecordingSpectrophotometer UV-240 (Shimadzu, Co. Ltd., Japan) was used to measurethe growth and enzyme activity of the bacteria.

Activated charcoal, Dowex 1X1-100 (Cl—) and Dowex 50W-X4 (H⁺) werepurchased from Sigma Chemical Co. (St. Louis. 63178, USA); and Bio-gelP₂ gel for a gel filtration was purchased from Bio-Rad (USA). An animalderived adenosine deaminase was purchased from Sigma Chemical Co., whichare 250 units of freeze-dried enzyme.

Silica gel G-60 and TLC-RP18F₂₅₄ (Merck) were used as a TLC plate forexamining purities; Multiphore II (LKB 2117) for high voltage paperelectrophoresis; and Model 510 (Waters, USA) for HPLC, wherein A PAK™C₁₈ and UV detector 441 were used as column and a detector,respectively.

For structural analyses, FT-S-60 IR spectroscopy (Bio-Rad), NMR andGC-MASS (Hewlett-Packard) were used.

The following Examples and Test Examples are given for the purpose ofillustration only, and they should not be construed as limiting thescope of the present invention.

EXAMPLE Example 1 Isolation and Identification of Bacillus sp. IADA-7(KCTC 10446BP) Strain

I. Isolation of a Bacterial Strain Producing Adenosine DeaminaseInhibitor

To isolate a bacterial strain producing an adenosine deaminaseinhibitor, a soil sample collected from a certain area of Korea wasair-dried at a dark place for 2 to 3 days. After about 0.5 g of thedried soil sample was suspended in 5 mL of distilled water and stood fora while, a supernatant taken from the suspension was spread onto themedium for isolating Actinomycetes (Medium A) having the followingcomposition of Table 1 and cultured at 30° C.

TABLE 1 Medium for isolating Actinomycetes (Medium A) Soluble starch 1.0g KH₂PO₄ 0.05 g NH₄Cl 0.05 g Agar 1.7 g Distilled water 100 mL Mediumfor synthesizing an inhibitor (Medium B) Glucose 1.0 g Yeast extract 0.2g Meat extract 0.2 g Peptone 0.2 g KH₂PO₄ 0.05 g MgCl₂.6H₂O 0.01 gDistilled water 100 mL (pH 7.3) Medium for storage (Medium C) Glucose1.0 g Peptone 0.2 g Meat extract 0.1 g Yeast extract 0.1 g Agar 1.8 gDistilled water 100 mL (pH 7.5) Medium for main culture (Medium D)Glucose 0.5 g Peptone 0.5 g Yeast extract 1.0 g NH₄Cl 0.5 g DistilledWater 100 mL (pH 7.0)

Using the medium for isolating Actinomycetes as a medium for isolating abacterial strain was intended to efficiently isolate a new bacterialstrain by modifying an isolating condition. To examine whether purelyisolated bacterial strains synthesize an enzyme inhibitor or not, thebacterial strains were inoculated in a test tube (2.4×21 cm) containing10 mL of the medium for synthesizing an inhibitor (Medium B) describedin Table 1 and cultured at 30° C. for 24 hrs in a shaking water bath.

After the culture solution was subjected to centrifugation (10,000×g, 20min) to isolate a supernatant, the supernatant was used to examinewhether the enzyme inhibitor was synthesized or not. The isolated strainwas cultured in a medium for storage (Medium C) described in Table 1 andstored at a cold dark room until it was used in the followingexperiment.

II. Medium and Cultivation

To synthesize an enzyme inhibitor, the isolated strain was inoculatedinto a test tube (1.5×15 cm) containing 3 mL of Medium B (Table 1) witha platinum loop and pre-cultured in a shaking incubator (120 Rev.×6 cmstroke) at 30° C. for 16 hrs. The pre-cultured strain (100 μl) wasinoculated into a shaking flask (500 mL in volume) containing 100 mL ofthe medium for main culture (Medium B. Table 1) and cultured at 30° C.for 24 hrs in a shaking water bath. Then, the growth and inhibitorsynthesis of the bacterial strain were measured.

To prepare a crude enzyme solution of adenosine deaminase originatedfrom a microorganism, Nocardioides sp. J-326TK was inoculated in a testtube (1.5×15 cm) containing 3 mL of Medium D (Table 1) with a platinumloop and pre-cultured in a shaking incubator (120 Rev.×6 cm stroke) at30° C. for 24 hrs. The pre-cultured strains (100 μl) was inoculated in ashaking flask (500 mL in volume) containing 100 mL of Medium D (Table 1)and cultured at 30° C. for 18 hrs in a shaking water bath. Then, theculture solution was subjected to centrifugation (10,000×g, 4° C., 20min) to isolate a supernatant and the supernatant was used as a crudeenzyme solution.

Test Example 1 Preparation of Enzyme Solution and Measurement of EnzymeActivity

I. Methods for Preparing an Enzyme Solution and Measuring an EnzymeActivity

An animal-derived adenosine deaminase used in this experiment was anenzyme extracted from a bovine pancreas followed by freeze-drying.First, the freeze-dried enzyme was dissolved in 3 mL of a potassiumphosphate buffer (pH 7.0) to prepare an enzyme solution. 50 μl of theenzyme solution was collected and diluted with the same buffer in aproper ratio and used as an enzyme for an enzyme reaction. Further, amicroorganism-derived enzyme was prepared by culturing Nocardioides sp.J-326TK in Medium D (Table 1) and centrifuging the culture solution toseparate a supernatant. The supernatant thus obtained was used as acrude enzyme solution.

An enzyme activity was measured according to the method described by theKalckar. Namely, a reaction mixture (final volume: 1.0 mL) comprising 5μmoles of adenosine, 50 μmoles of a potassium phosphate buffer (pH 7.0)and a proper amount of the enzyme solution were reacted in a water bathat 37° C. for 30 min and then subjected to boiling at 100° C. for 4 minto stop the enzyme reaction. The reaction mixture was diluted 100-foldwith the same buffer and its absorbance was measured at 265 nm.

1 Unit of enzyme activity was defined as the amount of an enzymesynthesizing 1 μmole of inosine at the above-mentioned condition for 1hr.

II. Measurement of Inhibitory Activity of an Adenosine DeaminaseInhibitor

An inhibitory activity of an inhibitor compound was determined by usingan inhibition rate which inhibits a deamination reaction of adenosinedeamidase. Namely, the inhibition rate was calculated from thedifference between an enzyme activity of a standard control and anenzyme activity when an inhibitor was treated. The amount of enzyme forthe measurement of an enzyme activity was about 17˜26 units/ml, and theconcentration of an inhibitor compound was regulated so that theinhibition rate of an adenosine deaminase activity can be 70% or below.

The inhibitory activity of 1 mL sample showing 50% inhibition rate wasdefined as 1 unit. The inhibition rate and inhibitory activity werecalculated as follows.Inhibition rate=(enzyme activity−sample activity)/enzyme activity×100(%)Inhibitory activity=inhibition rate/50×dilution rate (units/mL)

Example 2 Purification of an Adenosine Deaminase Inhibitor

To purify an adenosine deaminase inhibitor, after the culturesupernatant was separated by centrifugation, pre-treated with anactivated charcoal and extracted with 80% methanol, the extract obtainedthus was concentrated under a reduced pressure. The concentrated extractwas successively subjected to methanol fraction, Dowex 1X1-100 (Cl—)column chromatography and Dowex 50W-X4 (H⁺) column chromatography, tocollect fractions showing an inhibitory activity. The collectedfractions were combined and subjected to Bio gel P2 gel filtration,normal phase TLC and reverse phase TLC, in order.

Test Example 2 Classification and Identification of the Isolated Strain

Morphological, cultural and biochemical features of the isolated strainIADA-7 (KCTC 10446BP) were examined to characterize its taxonomicalposition, and a classification and identification of the isolated strainIADA-7 (KCTC 10446BP) were carried out according to the methodsdescribed in “Bergey's Manual of Bacteriology” (the 8^(th) Edition) and“Bergey's Manual of Systematic Bacteriology” (Vol. 2).

1) Morphological Feature

A morphological experiment of the isolated strain was carried outaccording to the methods described in “Laboratory Microbiology” (the3^(rd) Edition), “Classification and identification of microorganism”and “Manual of Methods for General Bacteriology”.

The isolated strain IADA-7 (KCTC 10446BP) was inoculated in a nutrientbroth agar medium and cultured for 5, 10, 20 and 48 hrs to observe itsmorphological change according to time course. As shown in Table 2 andFIG. 1 (a length of bar in FIG. 1 is 5 μm), it was found that theisolated strain IADA-7 is a Gram-negative, rod-shaped strain which has amotility and forms a spore.

TABLE 2 Article Feature Morphology Rod shape with round end MotilityMotile Gram staining Positive Spore Spore forming2) Cultural Feature

A gelatin stab medium experiment was performed using a nutrient gelatinstab medium, and a carbohydrate fermentation experiment was carried outaccording to the method described in “Biochemical Tests forIdentification of Medical Bacteria”.

As shown in Table 3, the cultural features of the isolated strain IADA-7(KCTC 10446BP) were characterized that it used glucose, fructose,D-arabinose, maltose and trehalose as a carbon source, but did not usemannitol, xylose, L-arabinose and lactose.

TABLE 3 Article Isolated strain (IADA-7) D-glucose + D-mannitol −D-fructose + D-xylose − D-arabinose + L-arabinose − L-xylose − Lactose −Maltose + Trehalose + Sorbitol − Inoline − Saccharose − +: use, −:nonuse3) Biochemical Feature

The biochemical feature of the isolated strain was analyzed according tothe methods described in “Biochemical Tests for Identification ofMedical Bacteria” and “Manual of Methods for General Bacteriology”.

As a result of examining the biochemical feature of the isolated strainIADA-7 (KCTC 10446BP), as shown in Table 4, it was found that it showedpositive signals in a catalase test, gelatin liquefaction and ureasetest, but a negative signal in a lysine decarboxylase test.

TABLE 4 Article Isolated strain (IADA-7) Catalse test + Anaerobic growth− Indol test − Gelatin liquefaction + Lysine decarboxylase − Ornitinedecarboxylase − Arginine decarboxylase + Urease test − H₂S production −4) Classification and Identification of the Isolated Strain

According to the “Bergey's Manual of Systematic Bacteriology” (Vol. 2),most of chemoheterotrophic and catalase-positive strains are belongs toBacillus sp. As described in the above, the isolated strain IADA-7 (KCTC10446BP) which was Gram-positive, aerobic, endospore forming rod-shapedstrain was identified as Bacillus sp. by examining its cultural andbiochemical features and comparing them with those of “Bergey's Manualof Bacteriology” (the 8^(th) Edition) and “Bergey's Manual of SystematicBacteriology” (Vol. 2).

Consequently, the isolated strain IADA-7 was designated Bacillus sp.IADA-7 and deposited at Korean Collection for Type Cultures (Address:#52, Oun-dong, Yusong-ku, Taejon 305-333, Republic of Korea) on Mar. 18,2003 under the accession number of KCTC 10446BP, in accordance with theterms of the Budapest Treaty on the International Recognition of theDeposit of Microorganism for the Purpose of Patent Procedure.

Example 3 Examination of a Synthetic Condition of an Adenosine DeaminaseInhibitor

1) Effects of the Amount of Ventilation and Initial pH

The effects of pH and the amount of ventilation on a cell growth andsynthesis of an inhibitor compound were examined. As a result, as shownin Table 5, it was found that when it was well ventilated, the cellgrowth and synthesis of an inhibitor compound were increased. Further,as shown in Table 6, the cell growth was favorable in an alkali pH rangerather than an acidic pH range and the inhibitor compound was alsosynthesized more in the alkali pH range.

TABLE 5 The amount of medium (mL) Activity (units/mL) 50 4,600 100 4,800150 4,300 200 4,000 250 3,600 The medium is composed of glucose 1.0 g,yeast extract 0.2 g, meat extract 0.2 g, peptone 0.2 g, KH₂PO₄ 0.05 gand MgCl₂.6H₂O 0.01 g.

TABLE 6 Initial pH Activity (units/mL) 3 400 6 4,300 7 4,900 9 4,600 Themedium is composed of glucose 1.0 g, yeast extract 0.2 g, meat extract0.2 g, peptone 0.2 g, KH₂PO₄ 0.05 g and MgCl₂.6H₂O 0.01 g2) Cell Growth and Synthesis of an Inhibitor

Cell growth, synthesis of an inhibitor compound and pH variation weremeasured according to time course of cultivation. As a result, as shownin FIG. 2, it was found that the cell growth was the highest at 24 hrsafter the cultivation and the synthesis of an inhibitor compound becameslightly decreased after 24 hrs (in FIG. 2, ‘□’ means pH, ‘∘’ means acell growth, and ‘●’ inhibitory activity).

Example 4 Purification of an Adenosine Deaminase Inhibitor

1) Preparation of a Supernatant

After the isolated strain was picked with a platinum loop from a storageslant culture tube and inoculated in a test tube containing 3 mL of amedium for synthesizing an inhibitor, the test tube was incubated at 30°C. for 16 hrs in a shaking water bath. The culture solution (100 μl) wastransferred to 500 mL in volume of a shaking flask containing 100 mL ofthe same medium and cultured at 30° C. for 24 hrs in a shaking waterbath.

The culture solution was subjected to centrifugation (10,000×g, 4° C.,20 min) to remove cells, and a supernatant thus obtained was used as acrude inhibitor solution of the adenosine deaminase. Since thesupernatant showed 5,100 units per 1 mL of an enzyme activity and atotal volume of the supernatant was 8,820 mL, a total activity was45,000,000 units.

2) Extraction with an Activated Charcoal

After pH of the culture solution (300 mL, 4,500 units/mL, 1,350,000units) was adjusted to 7.0, 6 g of a dried activated charcoal washedwith methanol was added thereto and the reaction mixture was stirred.

After the reaction mixture was subjected to aspiration for 10 min, theactivated charcoal on a filter paper was washed with distilled water.The washed activated charcoal was divided into 6 equal parts (225,000units) and extracted with six kinds of organic solvents by aspiration,respectively (Table 7). As shown in Table 7, since when the activatedcharcoal was extracted with 80% methanol, the extraction yield was thehighest, 200 mL of the concentrated supernatant (7,740,000 units) wasdivided into 4 equal parts (50 mL; 1,935,000 units) and pH of eachsupernatant was adjusted to 2, 6, 7 and 8, respectively. Then, eachsupernatant was subjected to absorption to the activated charcoal andextracted with 80% methanol (Table 8).

TABLE 7 Inhibitory activity Solvent (units) Yield (%)  80% methanol126,000 56 100% ethanol 112,500 50  80% butanol 87,000 39 100% hexanol81,000 36  50% chloroform 69,750 31  50% acetone 74,200 33

TABLE 8 Inhibitory Solvent pH activity (units) Yield (%) Methanol (80%)2 619,000 32 6 1,199,700 62 7 1,393,200 72 8 1,470,000 76

According to the results shown in Tables 7 and 8, after pH of thesupernatant was adjusted to 8 during the purification, the activatedcharcoal (Sigma, 100˜400 mesh) was added to the culture solution at afinal volume of 2 w/v/% and the reaction mixture was stirred for 10 min.Then, the reaction mixture was subjected to aspiration using anaspirator. After the aspiration, the reaction mixture was washed withthe tertiary distilled water twice in a quarter volume of the culturesolution and was extracted with the equal volume of 80% methanol to theculture solution four times. The extract was concentrated into a finalvolume of 100 mL under a reduced pressure and subjected tocentrifugation (10,000×g, 30 min) and filtration using a membrane filterhaving 2 μm in pore size (Disposable sterile syringe filter) to removethe activated charcoal.

Since the activated charcoal extract showed 310,500 units of an enzymeactivity per 1 mL and a total volume of the concentrated activatedcharcoal extract was 100 mL, a total activity was 31,050,000 units andthe yield for the supernatant was 69%.

3) Methanol Fractionation

Cold methanol was added to the collected sample extracted with theactivated charcoal at a final volume of 50%, and the mixture was kept ata freezer at −20° C. for 24 hrs. After the frozen mixture was subjectedto centrifugation (10,000×g, 30 min) to remove a precipitate andmethanol was evaporated with a rotary evaporator at 45° C., distilledwater was added thereto and its pH was adjusted to 8.0 with 1 N NaOH.

Since the methanol fraction showed 558,000 units of an enzyme activityper 1 mL and a total volume of the methanol fraction supernatant was 50mL, a total activity was 27,900,000 units and the yield for thesupernatant was 62%.

4) Dowex 1X1-100 (Cl—) Ion Exclusive Chromatography

After confirming (+) electric charge with high voltage paperelectrophoresis, an ion exclusive chromatography was carried out. Themethanol fraction was loaded onto Dowex 1X1-100 (Cl—) column (3.4×37 cm)at a flow rate of 0.25 mL/min and the column was washed with distilledwater. Since the concentrate obtained from the Dowex 1X1-100 (Cl—)column showed 462,000 units of an enzyme activity per 1 mL and a totalvolume of the concentrate was 50 mL, a total activity was 23,100,000units and the yield for the supernatant was 51%.

5) Dowex 50W-X4 (H⁺) Ion Exchange Chromatography

The sample obtained from the Dowex 1X1-100 (Cl—) column was loaded ontoDowex 50W-X4 (H⁺) column (3.4×37 cm) at a flow rate of 0.25 mL/min andthe column was washed with the tertiary distilled water. An inhibitorcompound absorbed to the column was eluted with 0.2 N NH₄OH at a flowrate of 0.25 mL/min (FIG. 3). An effluent was separately distributed by30 mL, concentrated under a reduced pressure, and then, its activity wasmeasured.

As shown in FIG. 3, fractions 1 to 3 and after 8 were discarded becausethey showed a tailing and low productivity of the inhibitor compound,and fractions 4 to 7 were concentrated under a reduced pressure to make30 mL of an effluent.

Since the concentrate of fractions 4 to 7 obtained from the Dowex 50W-X4(H⁺) ion exchange chromatography showed 37,000 units of an enzymeactivity per 1 mL and a total volume of the concentrate was 30 mL, atotal activity was 7,100,000 units and the yield for the supernatant was15%.

6) Bio-Gel P₂ Gel Filtration

Fractions 4 to 7 obtained from Dowex 50W-X4 (H+) ion exchangechromatography were collected and concentrated into 2 mL under a reducedpressure. The concentrate was loaded onto Bio-gel P₂ column (1.6×48 cm)and fractionated by 3 mL with the tertiary distilled water at a flowrate of 0.18 mL/min. Elution profiles of each sample are shown in FIG.4.

Since the concentrate of fractions 35 to 39 obtained from the gelfiltration showed 225,000 units of an enzyme activity per 1 mL and atotal volume of the concentrate was 10 mL, a total activity was2,250,000 units and the yield for the supernatant was 5%.

7) Reverse Phase TLC

Thin layer chromatography (TLC) has the advantages over paperchromatography that it can save the time for developing, efficientlycarry out the development, and detect a very small amount of a compoundbecause the compound becomes concentrated onto a spot. Since normalphase TLC employs a polar substance as a stationary phase, it has ademerit that a tailing is often occurred when the polar substance isloaded thereon. Therefore, the present invention used reverse phase TLCwhich uses a non-polar substance as a stationary phase.

Fractions 35 to 39 obtained from Bio-gel P₂ gel filtration werecollected and concentrated into 2 mL under a reduced pressure. Theconcentrated sample was dropped on a reverse phase-18F₂₅₄ plate (20×20cm) and developed in an solvent system (NH₄Cl:ethanol:water=6:4:1(v/v/v)).

The development of an inhibitor compound was examined under UVirradiation. Then, silica gels positioned at 0.3 cm up and down fromR_(f)=0.85 were scratched from the paper and eluted with methanol. Theextract was concentrated under a reduced pressure to remove methanol,and then, the concentrated sample was dissolved in 2 mL of HPLC water.

8) Normal Phase TLC

The sample (2 mL) obtained from reverse phase TLC was dropped on asilica gel G-60 plate (20×20 cm) and developed in an solvent system(chloroform:methanol=7:3, v/v). To confirm the development of aninhibitor compound, a piece of paper severed from left and right ends ofthe developing paper by 2 cm was steamed with iodine vapor. Then, silicagels positioned at 0.3 cm up and down from R_(f)=0.35 were scratchedfrom the paper and eluted with methanol. The extract was concentratedunder a reduced pressure to remove methanol, and then, the concentratedsample was dissolved in 2 mL of HPLC water.

Generally, when the silica gel scratched from TLC is eluted, the silicagel remains in an effluent, which makes difficult a completepurification. To solve this problem, the present invention carried outcentrifugation (10,000×g, 30 min) and filtration using a membrane fillerhaving 0.2 μm in pore size. After sufficiently packing a cotton wool ina column (1×7 cm) at first, Dowex 1X1-100 (Cl—) resin was packed thereonand eluted with 0.2 N ammonium solution at a flow rate of 0.25 mL/min.Then, the eluted sample was freeze-dried.

The overall procedure was summarized in FIG. 5. An inhibitor compoundproduced by Bacillus sp. IADA-7 (KCTC 10446BP) was purified from theculture solution in a yield of about 2% which corresponds to a dryweight of 9.6 mg.

Experimental Example 3 Confirmation of Purity of an Adenosine DeaminaseInhibitor

1) Normal Phase TLC

The purified sample was loaded onto a silica gel G-60 plate to examineits purity and developed in an solvent system (chloroform:methanol=7:3,v/v). A single spot was detected by steaming with iodine vapor and itsresult was shown in FIG. 6. As shown in FIG. 6, the single spot wasdetected at a position of R_(f)=0.35.

2) Reverse Phase TLC

The purified sample was loaded onto a reverse phase-18F₂₅₄ plate anddeveloped in an solvent system (NH₄Cl:ethanol:water=6:4:1 (v/v/v)).After a spot was detected by UV, a single spot was detected by steamingwith iodine vapor and its result was shown in FIG. 7. As shown in FIG.7, the single spot was detected at a position of R_(f)=0.85.

3) High Voltage Paper Electrophoresis

Since when a micromolecule substance is isolated by low voltage paperelectrophoresis, diffusion occurs too fast, the present invention hasused high voltage paper electrophoresis. However, since heat isgenerated due to high voltage, it is positively necessary to use acooling system.

Paper electrophoresis was carried out according to the method describedby Teintze et al.

After a paper electrophoresis kit was filled with a potassium phosphatebuffer (pH 7.0, 0.02 M), the both ends of Whatman No. 1 paper weresoaked in the buffer for 7 hrs to be saturated. The purified sample (20μl, 90 units/mL) was spotted at a start point of the saturated paper andsubjected to electrophoresis at 300 V, 40 A. Then, after drying thepaper, it was steamed with iodine vapor to detect a single spot. Theresult was shown in FIG. 8. As a result of presenting the extent of anelectric charge as an absolute value by using bromophenol blue (BPB) asa control, R_(f) value was 0.13.

4) HPLC (High Performance Liquid Chromatography)

It has been recently developed a hard gel type of carriers such as finepolyvinyl-based lipophilic polymers, porous silica and porous polymers,which show a fast flow rate and resistance to high pressure and haveabout 10 μm in diameter of a gel particle, and used for a HPLCpurification.

Since when a polar substance is isolated using a reverse phase column,its separation efficiency increases, the present invention used Novapak™ C₁₈ column (Waters).

The purified sample which was detected as a single spot in normal phaseTLC, reverse phase TLC and high voltage paper electrophoresis wassubjected to HPLC using Nova pak™C₁₈ column. At this time, a flow ratewas 0.8 mL/min, HPLC water was used as a solvent, and UV detector 441was used as a detector. As shown in FIG. 9, a sharp single peak wasdetected at RT=8.5 min. FIG. 10 showed the relationship between theamount of the purified sample injected and a height of each peak.

As shown in FIG. 10, a linear relationship was shown between theinjection amount and a peak's height, which makes possible ofquantitatively analyzing by measuring the peak's height.

As shown in the above, since the purified sample was detected as asingle spot in normal phase TLC, reverse phase TLC and high voltagepaper electrophoresis, and as a single peak in HPLC analysis, it wasregarded that the sample was completely purified, and therefore, thesample was freeze-dried and subjected to the following structuralanalysis.

Test Example 4 Measurement of a Molecular Weight of an AdenosineDeaminase Inhibitor

To measure a molecular weight of the sample purified in the above step,the sample was subjected to gel filtration using Bio-gel P₂ column(1.6×48 cm). Vitamin B₁₂ (M.W. 1,335), BPB (M.W. 669) and cytosine (M.W.110) were used as a standard control, and the result was shown in FIG.11. The ratio (Vo/Ve) of an elution volume to a void volume (Vo) wasplotted to a logarithmic value of a molecular weight of the standardcontrol. The void volume was determined as the elution volume ofhemoglobin (M.W. 64,000).

Vo/Ve of vitamin B₁₂ (M.W. 1,335) was 3.3, that of BPB (M.W. 669) was2.6, that of cytosine (M.W. 110) was 3.3, and that of the inhibitorcompound of the present invention was 3.1. Accordingly, the molecularweight of the inhibitor compound was determined as about 175.

Experimental Example 1 Inhibition Mode of an Adenosine DeaminaseInhibitor

The effect of an adenosine concentration on adenosine deaminasespurified from bovine pancreas and Nocardiodes sp. J-326TK cell,respectively, was measured from a Lineweaver and Burk plot. As shown inFIGS. 12 and 13, Km value of the animal-derived enzyme was 0.027 mM, andthat of the microorganism-derived enzyme was 0.2 mM. Ki values of 0.01and 0.02 mg of the purified inhibitor compound to the animal- andmicroorganism-derived enzymes, respectively, were also measured from aLineweaver and Burk plot. As shown in FIGS. 12 and 13, Ki value of theanimal-derived enzyme was 0.027 mM, and that of themicroorganism-derived enzyme was 0.2 mM. (in FIGS. 12 and 13, -∘-, --●--and -●- represents 0, 0.01 and 0.02 mg of the inhibitor compound,respectively).

Like the results described above, since Km value was identical to Kivalue and Vmax value decreased as the concentration of the inhibitorcompound increased from 0.01 mg to 0.02 mg.

Test Example 5 Physicochemical Property of an Adenosine DeaminaseInhibitor

1) Stability Test

As a result of measuring a residual activity after pH of the culturesolution was adjusted to 2, 7 and 9, respectively, and kept at 70° C.for 30 min, it was stable in an alkali pH range (Table 9). Further, as aresult of examining the condition for concentrating the culture solutionusing a rotary vacuum evaporator, it was found that the inhibitorcompound of the present invention didn't lose its activity whensubjected to the concentration at 45° C. for a long time.

TABLE 9 Incubation Residual activity (units/mL) Relative activity (%) pH2 2,900 7 pH 7 19,500 93 pH 9 21,000 100

From the results described in the above, it was found that the inhibitorcompound of the present invention was very stable around at roomtemperature. Accordingly, the purified sample was adjusted its pH to 8and stored at low temperature of about 5° C.

2) UV Absorption Spectrum

As shown in FIG. 14, UV absorption spectrum of the purified sampledissolved in milipore water showed the maximum peak at 232 nm and didn'tshow any peak at a visible range (in FIG. 14, a horizontal linerepresents OD value and the vertical line represents an absorbance(nm)).

Since IR spectrum 1669 cm⁻¹ showed the absorbance of an acetamide andC═O was observed at 192 ppm of ¹³C-NMR spectrum, it was predicted thatthe inhibitor compound of the present invention has the acetamide group.Since the acetamide shows the maximum peak at 220 nm, and when acarbonyl-containing compound bound to the acetamide, the maximum peakmay be changed (transition) into 232 nm, it was estimated that the IRspectrum was identical to the NMR spectrum.

3) IR Spectrum

A molecule has a unique oscillation frequency. When the molecule isapplied infrared radiation with successively changing a wavelength, theinfrared radiation equal to the unique oscillation frequency of themolecule is absorbed, and accordingly, a spectrum according to themolecular structure is obtained. To analyze the molecular structure fromthe spectrum is an infrared absorption spectroscopy method.

The purified sample (2 mg) was sufficiently ground four times withadding 50 mg of KBr powder per once (total 200 mg). This grinding stepwas carried out in a dry box to prevent from absorbing water. The groundsample was transferred to an oil pressure processor and slowlypressurized to make a disk. The disk was subjected to FT-IR at 8 cm⁻¹ ofa resolution and 32 scans of a scanning number, and the result was shownin FIG. 15.

As shown in FIG. 15, the adenosine deaminase inhibitor compound of thepresent invention showed an acetamide peak at 1669 cm⁻¹ and a pyrol peakat 3037 cm⁻¹.

4) ¹H-NMR Spectrum

Two types of spins, i.e., +½ and −½H, are existed in an atomic isnucleus of H. When this nucleus is put on a magnetic field, each spinoriginates toward a different energy level. When electromagnetic waveshaving a resonance frequency are irradiated thereto, +½ type of the spinchanges into −½ type of the spin due to energy absorption. To find amolecular structure by detecting this frequency is a NMR spectrummethod.

The purified sample (2 mg) dissolved in D₂O was filled in a test tube upto 38 mm and subjected to NMR analysis. 500 MHz NMR spectrum was shownin FIG. 16.

5) ¹³C-NMR Spectrum

When ¹³C nucleus is put on a magnetic field, it divides into +½ and −½of energy levels and +½ type of the low energy level more exists than −½type at a thermal equilibrium state. When electromagnetic waves having aresonance frequency are irradiated thereto, +½ type of the nucleuschanges into −½ type of the nucleus. When +½ type of the nucleus becomesdecreased due to a continuous irradiation, and finally, the number of +½type of the nucleus is equal to that of −½ type of the nucleus, anabsorption does not occurred further and this state is called asaturation.

The spectrum obtained by dissolving 10 mg of the purified sample in D₂Oand filling in a microcell capillary of 10 mm in outside diameter and180 mm in length was shown in FIG. 17. Further, ¹³C-H NMR spectrum wasshown in FIG. 18.

6) GC-MASS Spectrum

When a sample molecule vaporized by heating in a high-degree vacuum isapplied a large energy such as an electron flow, one electron isdeviated from the molecule, which results in generating a cationicradical of the molecule (M⁺, molecular ion). This radical is openedagain, to generate several ions called a fragment ion. An equipment forseparating and recording these ions according to the ratio (m/e) of amass (m) and an electric charge (e) is a quantitative analyzer, and thespectrum obtained therefrom is called MASS spectrum. There are two kindsof MASS, i.e., EI-MASS and CI-MASS. The present invention used EI-MASS.

The spectrum obtained by dissolving the purified sample in D₂O was shownin FIG. 19, and the molecular weight of the inhibitor compound of thepresent invention was measured as 124.

7) Structural Analysis

As a result of analyzing the inhibitor compound of the present inventionwith UV absorption spectrum, IR spectrum, ¹H-NMR spectrum, ¹³C-NMRspectrum, ¹³C-¹H NMR spectrum and GC-MASS spectrum, it was possible topredict the structure of the inhibitor compound into three types asshown in FIG. 20.

An acetamide group was detected at IR spectrum 1669 cm⁻¹ and C═O wasobserved at ¹³C-NMR spectrum 192 ppm, which means that the inhibitorcompound contained the acetamide group. Since UVmax of the acetamidegroup was 220 nm, and when the other molecule bound thereto, the UVmaxmay be changed into 232 nm, IR spectrum was identical to UV spectrum.Further, the result of comparing ¹³C-¹H NMR spectrum was shown in Table10.

TABLE 10 ¹³C-¹H NMR spectrum Analytic result 12-18 CH₂ (ppm) 23-23 CH₂34-38 CH₃ 57 CH₂ 59 —C— 75 impurities 192  C═O

As shown in Table 10, it was found that the inhibitor compound of thepresent invention was a heterocyclic compound.

Experimental Example 2 Antibacterial Activity Test

To test an antibacterial activity of the inhibitor compound of thepresent invention, Gram-positive strain Staphylococcus aureus andGram-negative strain Escherichia coli were cultured at 37° C. incubatorfor 18 hrs, 100 μl of each culture solution was densely spread onto a LBagar plate with a platinum loop, and then, cultured at 37° C. incubatorfor 18 hrs.

When a disc was put on the surface of each incubated plate and 100 unitsof the inhibitor compound of the present invention was inoculated to thedisc, antibacterial rings of 11 mm and 15 mm in diameter was formed atthe both plates of Staphylococcus aureus and Escherichia coli,respectively.

Experimental Example 3 Cytotoxicity of Inhibitor to Human Cancer Cell(1)

Cytotoxicity was measured according to modified MTT assay described byMosmann with a proper modification. Human transitional-cell carcinomaJ82 cells derived from a testis (bladder) were suspended in a serum-freeRPMI 1640 medium and PBS (phosphate-buffered saline; KCl 0.2 g, KH₂PO₄0.2 g, NaCl 8.0 g, Na₂HPO₄ 1.15 g, MgCl₂.6H₂O 0.101 g/L, pH7.4) anddistributed at a well plate in the amount of 100 μl per well. Theconcentration of the purified inhibitor compound was adjusted to 0.1, 1and 10 μg/mL, respectively, and 20 μl each of the inhibitor compound wasadded to the well plate. At this time, the equal volume of water wasused as a control. The well plate was incubated at 37° C., 5% CO₂ for 3days, and the extent of cytotoxicity was determined by using anabsorbance measured at 650 nm. The result was shown in FIG. 21.

As shown in FIG. 21, OD values of each plate added with water (control),0.1, 1 and 10 μg/mL of inhibitor were 1.100, 0.950, 0.919 and 0.893,respectively. Accordingly, the inhibitor compound of the presentinvention showed about 19% of cytotoxicity at a concentration of 10μg/mL.

Experimental Example 4 Cytotoxicity of Inhibitor to Human Cancer Cell(2)

Cytotoxicity was measured according to MTT assay described by Mosmann,and a human leukemia cancer cell, Jurkat T cell was used as a targetcell.

When the concentration of the inhibitor compound of the presentinvention was 40 μg/mL, 50% of cells died, thus showing that IC₅₀ of theinhibitor compound is about 40 μg/mL.

From the results described in the above, it was found that the compoundof Formula 1, which was isolated from the novel Bacillus sp. IADA-7strain, can be effectively used as an adenosine deaminase inhibitorhaving high antibacterial and anticancer activities.

While the embodiments of the subject invention have been described andillustrated, it is obvious that various changes and modifications can bemade therein without departing from the spirit of the present inventionwhich should be limited only by the scope of the appended claims.

1. A compound of Formula 1 and a pharmaceutically acceptable saltthereof:

wherein R₁ is H or C₁-C₁₀ alkyl; and R₂ is H or C₁-C₁₀ alkyl.
 2. Apharmaceutical composition comprising the compound of claim 1 and apharmaceutically acceptable carrier.
 3. A compound of claim 1, whereinR₁ is H or CH₃; and R₂ is H or CH₃.
 4. A pharmaceutical compositioncomprising the compound of claim 3 and a pharmaceutically acceptablecarrier.